ChIC/CUT&RUN-seq protocol

Last edited Tue 26 June 2025

Chromatin Immuno-Cleavage/Cleavage Under Targets and Release Using Nuclease (ChIC/CUT&RUN) is a chromatin profiling technique used to analyze DNA-protein interactions. While ChIP-seq uses sonication of fixed cells to fragment chromatin, ChIC/CUT&RUN-seq uses an enzyme (pAG-MNase).

The pAG-MNase enzyme is conjugated to an antibody for proteins of interest. The fusion protein is encoded by a specifically designed coding sequence to ensure optimal expression and purification. A schematic diagram of the fusion protein structure, including its functional domains and tags, is often used to illustrate these modifications. Chromatin fragments containing proteins of interest can then be purified using immunoprecipitation techniques. After this, the DNA fragments are purified and sequenced. The sequencing results can be used to determine the regions of DNA with which your protein of interest interacts. E. coli DNA introduced with the fusion protein serves as a spike-in control for normalization. A calibration strategy based on the carry-over of E. coli DNA allows for accurate normalization and comparison across samples.

Introduction to CUT&RUN

CUT&RUN is a cutting-edge technique that has transformed the study of gene expression and the mechanisms regulating gene expression at the chromatin level. Unlike traditional methods, CUT&RUN enables researchers to profile protein-DNA interactions with remarkable sensitivity, even from a small number of cells. The method leverages a fusion protein, Protein A-Protein-G Micrococcal Nuclease (pAG-MNase), which is directed to specific chromatin regions by antibodies against a target transcription factor or chromatin-associated protein. Upon activation, the pAG-MNase cleaves DNA near the binding site, releasing cleaved fragments into the supernatant. These DNA fragments are then extracted and sequenced, providing a high-resolution map of protein-DNA interactions that are crucial for understanding gene regulation in molecular biology.

Background and principles

The core principle of CUT&RUN is the targeted cleavage of chromatin in unfixed, permeabilized cells. The process begins with the incubation of cells with a primary antibody that specifically binds to a DNA-associated protein, such as a transcription factor. To expand antibody compatibility, a fusion protein, either Protein A-MNase or a hybrid Protein A-Protein G-MNase, is introduced, allowing for the recognition of a wide range of antibody species. Once the fusion or hybrid protein is tethered to the antibody-bound protein, the addition of calcium ions activates the MNase, which cleaves the DNA in close proximity to the protein of interest. The resulting cleaved fragments are released into the supernatant, ready for DNA extraction and sequencing. This streamlined approach supports routine epigenomic profiling, offering high efficiency, reproducibility, and scalability for high-throughput studies, all while maintaining compatibility with a variety of antibodies and cell types.

Stage 1 - Cell harvesting and bead preparation

Materials required

• ConA magnetic beads (10 µL per reaction)
• PBS
• Cell sample (2.5 x 10⁵ per reaction)
• Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail)
• Binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl₂ , 1 mM MnCl₂)
• Centrifuge
• Magnetic rack

Steps

Harvest cells and suspend in 3–4 mL ice-cold PBS.

Count cells and take the desired number of cells per reaction.

• We use 2.5x10⁵ HeLa cells per reaction. However, this may need optimizing for each cell line.
• See our counting cells using a hemocytometer protocol.

Prepare wash buffer.

Add protease inhibitor cocktail (eg ab65621) before use.

Wash cells three times in wash buffer.

• Add 1 mL of wash buffer to cells.
• Spin down cells at 600 x g for 3 min.
• Remove supernatant and repeat for a total of three washes.

Prepare a slurry of conA magnetic beads in binding buffer.

• Take 10 µL of beads per reaction.
• Wash beads in 1 mL of binding buffer on the magnetic rack.
• Resuspend the beads in 10 µL per reaction of binding buffer.

Bind cells to activated beads.

• Mix cells and beads together for 20 min at room temperature, mixing gently every 4 min.

Separate the cell-bound beads from solution.

• Place the tube on a magnetic rack for 1 – 2 min.
• Discard supernatant and keep beads.

Stage 2 - Permeabilization and binding of antibodies

Materials required

• Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail)
• 0.5 M EDTA stock solution
• 5% Digitonin stock solution
• Antibody buffer (see Step 1)
• Antibodies validated for ChIC/CUT&RUN-seq
• Horizontal gyratory shaker
• Magnetic rack

Steps

Prepare the antibody buffer.

• Dilute EDTA and Digitonin stock solutions in the Wash Buffer to include final concentrations of 0.2 mM EDTA and 0.05% Digitonin.
• Before use, add protease inhibitor cocktail (ab65621) and Digitonin (ab141501).

Resuspend beads in antibody buffer.

• Resuspend in around 100 µL per reaction and aliquot out into separate tubes if prepping serval reaction in same tube.

Add antibody to bead mix.

• Recommended dilutions will always be suggested on the antibody datasheet
• Incubate overnight at 4°C in a horizontal shaker.

Stage 3 - Binding of pAG-MNase

Materials required

• pAG-MNase
• Wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail, 0.05% Digitonin)
• 5% Digitonin stock solution
• Digitonin wash buffer (see step 2)
• Horizontal gyratory shaker
• Magnetic rack

Steps

Separate the antibody-bound beads from the solution.

• Place the tube on a magnetic rack for 1 – 2 min.
• Discard supernatant and keep beads.

Prepare Digitonin wash buffer.

• Dilute Digitonin stock solution in the wash buffer to give a final concentration of 0.05% Digitonin.
• Before use, add a protease inhibitor cocktail (ab65621) and Digitonin (ab141501).

Wash beads twice with Digitonin wash buffer.

• Add 200 µL of Digitonin wash buffer to the beads per reaction
• Place the tube on the magnetic rack for 1 min.
• Discard supernatant and keep beads.
• Repeat for a total of two washes.

Incubate the beads with pAG-MNase solution.

• Resuspend beads in 50 µL of digitonin wash buffer.
• Add pAG-MNase to a final concentration of 700 ng/µL.
• Incubate for 1 – 2 h at room temperature with horizontal shaking at 130 rpm.

Stage 4 - Chromatin-targeted digestion in CUT&RUN

Materials required

• Low salt buffer (20 mM HEPES pH 7.5, 0.05% Digitonin, 0,5 mM Spermidine)
• Incubation buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl₂, 0.05% Digitonin)
• Magnetic rack

Steps

Separate the antibody-bound beads from the solution.

• Place the tube on a magnetic rack for 1 – 2 min.
• Discard supernatant and keep beads.

Prepare low salt, incubation and wash buffers.

• Add Digitonin (ab141501) before use.
• Place buffers on ice.

Wash beads twice with Digitonin wash buffer.

• Add 200 µL of Digitonin wash buffer to the beads per reaction and incubate for 5 min.
• Place the tube on the magnetic rack for 1 min.
• Discard supernatant and keep beads.
• Repeat for a total of two washes.

Wash beads with low salt buffer.

• Add 200 µL of low salt buffer to the beads per reaction and incubate for 5 min.
• Place the tube on the magnetic rack for 1 min.
• Discard supernatant and keep beads.

Digest chromatin by incubating beads with ice-cold incubation buffer.

• Add 100 µL of incubation buffer and incubate for 15 min on ice, mixing halfway through the incubation.
• Place the tube on the magnetic rack for 1 min.
• Discard the supernatant and keep beads.
• The incubation buffer contains Ca²⁺ ions, which activate the pAG-MNase enzyme to cleave DNA. You may need to optimize the incubation time.

Stage 5 - Elution of DNA and sequencing

Materials required

• Stop buffer (20 mM EGTA, 170 mM NaCl, 0.05% Digitonin, 50 µg/mL RNAse A, 25 µg/mL Glycogen)
• Thermo-mixer
• Magnetic rack
• Commercial extraction, quality control, and library preparation kits

Steps

Incubate beads with stop buffer.

• Add 100 µL of ice-cold stop buffer.
• Incubate beads for 30 min at 37°C in a thermomixer, shaking at 700 rpm.

Collect fragments.

• Place the tube on the magnetic rack for 1 min.
• Aliquot the supernatant into new tubes.

Extract DNA and prepare for sequencing.

• Use commercial extraction, quality control, and library preparation kits according to the manufacturer’s instructions.

Send DNA fragments for sequencing.

Applications of CUT&RUN

CUT&RUN has rapidly become a versatile tool in molecular biology, enabling researchers to investigate a wide array of chromatin-associated phenomena. It is particularly powerful for mapping transcription factor binding sites, profiling histone modifications, and assessing chromatin accessibility across the genome. These applications are essential for unraveling the complexities of gene regulation, cell differentiation, and epigenetic modifications in diverse biological systems, including mammalian cells and plant cells. In the context of human disease, such as cancer, CUT&RUN allows for the detailed analysis of the epigenetic landscape, helping to identify key regulatory elements and potential therapeutic targets. Its ability to generate high-resolution data from limited cell numbers makes it ideal for studying rare cell populations and dynamic changes in gene expression.

Comparison to other methods

Compared to traditional chromatin immunoprecipitation (ChIP) techniques, CUT&RUN offers several significant advantages. It requires far fewer starting materials, making it suitable for experiments with limited cell samples or even single cells. The method provides higher sensitivity and specificity, reducing background noise and increasing the accuracy of detecting true protein-DNA interactions. Importantly, CUT&RUN eliminates the need for formaldehyde fixation, which can introduce cross-linking artifacts and bias the results. However, the technique does rely on the availability of high-quality, specific antibodies, and certain genomic regions may still present challenges in analysis. Overall, CUT&RUN represents a major advancement over ChIP for many applications in chromatin research.

Data processing and analysis

The analysis of CUT&RUN sequencing data involves a series of bioinformatic steps designed to extract meaningful insights into gene regulation and epigenetic modifications. After paired-end Illumina sequencing, reads are aligned to a reference genome to identify the precise locations of protein-DNA interactions. Peak calling algorithms are then used to pinpoint binding sites for transcription factors and regions of histone modifications. To ensure accurate quantification and account for variations in sequencing depth, spike-in DNA, such as E. coli DNA, can be added as an internal control. This calibration strategy helps normalize the data and avoid cross-mapping issues. Popular software tools like Bowtie2 and MACS2 facilitate data processing, while integration with other genomic and epigenomic datasets provides a comprehensive view of the regulatory landscape. This robust analytical framework enables researchers to uncover the molecular mechanisms underlying gene expression and chromatin dynamics.